Gingival fibroblasts are mesenchymal cells which are capable of migrating, adhering and proliferating within the soft connective tissues of the gum, thereby maintaining the integrity of the gingival tissue, which is exposed to numerous aggressions, such as mechanical stresses, bacterial infections, or pH and temperature variations. Gingival fibroblasts are in particular described in Gogly et al., (1997) Clin. Oral Invest. 1:147-152; Gogly et al. (1998) Biochem. Pharmacol. 56:1447-1454; and Ejeil et al. (2003) J. Periodontol. 74:188-195.
Depending on environmental conditions, gingival fibroblasts are capable to modulate their phenotype, and to respond by proliferating, migrating, synthesising matrix components or matrix-related enzymes. Gingival fibroblasts notably synthesise collagens (e.g. types I, III, V, VI, VII, XII) elastic fibers (oxytalan, elaunin and elastin), proteoglycans and glycosaminoglycans (e.g. decorin, biglycan), and glycoproteins (e.g. fibronectin, tenascin). Simultaneously, gingival fibroblasts synthesise enzymes that are able to degrade the macromolecular compounds (matrix metalloproteinases; MMPs), but also enzymes inhibiting active forms of MMPs (Inhibitors of metalloproteinases; TIMPs). Gingival fibroblasts are thus important actors of extracellular matrix remodelling.
Gingival fibroblasts have been shown to treat arterial-remodelling pathologies (WO 2006/013261) and more recently to promote and to accelerate skin wound healing.
Thus, the potential of gingival fibroblasts in cell-therapy appears promising and highlights the need for culture methods liable to yield large quantities of these cells.
Current culture media for gingival fibroblasts are well defined (Gogly et al. op. cit.). These culture media all make use of foetal calf serum (FCS). Indeed, it could be shown that gingival fibroblasts had very precise requirements in term of FCS concentration in the culture medium, since less than 10% FCS not only causes a decreased growth of gingival fibroblasts but also causes gingival fibroblasts to present a high lactate deshydrogenase (LDH) activity, which is indicative of cellular suffering (see FIGS. 1 and 2).
However, FCS, and animal sera in general, are unwanted in culture media used to cultivate and/or differentiate cells for human cell-therapy. Indeed, the composition of animal sera is usually variable and badly characterized (Morris & Warburton (1994) “Serum-screening and selection” in “Cell & Tissue Culture: Laboratory Procedures” (Doyle, Griffiths and Newell, eds) pp. 2B:101-105, Wiley). Moreover, these sera are liable to be contaminated by micro-organisms or prion and therefore are liable to be health-threatening to the patient for whom the cultivated cells are intended (Wessman & Levings (1999) Dev. Biol. Stand. 99:3-8).
Serum-free culture media for animal cells are known in the art and are commercially available. Thus, one may cite for instance the serum replacement formulation KNOCKOUT™ SR from GIBCO™ which is added to Dulbecco's Modified Eagle's Medium (DMEM), instead of FCS, to yield a serum-free culture medium. However, it has never been asserted that such a medium was liable to provide for adequate serum-free growth conditions of gingival fibroblasts, which, as recalled above, are known to have very specific requirements in term of FCS concentrations. Besides, these media have not been specifically designed to provide for gingival fibroblast culture conditions suited for therapeutic use, that is, culture conditions liable to rapidly yield large quantities of functional gingival fibroblasts.
Thus, it is an object of the present invention to improve existing serum-free culture media for animal or human cells in order to provide for optimal growth of gingival fibroblasts.